A novel assay for tracking carboxylesterase gene amplifications conferring

8 Laboratoire d’Ecologie Alpine (LECA), UMR 5553 CNRS – Université Grenoble-Alpes, 2233 rue de 9 la piscine, Grenoble, France. 10 United States Navy Entomology. Center of Excellence, NAS Jacksonville, Florida, United States of 11 America. 12 US Naval Medical Research Unit No. 2, Singapore. 13 Laboratoire Cogitamus, 1 3⁄4 rue Descartes, 75005 Paris, France. 14 Department of Microbiology, Khon Kaen University, Khon Kaen, Thailand. 15 Institut de Recherche pour le Développement, UMR IRD 224-CNRS 5290-Université Montpellier. 16 911, avenue Agropolis, BP 64501, 34394 Montpellier Cedex 5, FRANCE. 17 Medical and Veterinary Entomology, Institut Pasteur du Cambodge, P.O Box. 983 Phnom Penh, 18 Cambodia. 19 Medical Entomology and Vector-Borne Disease Laboratory, Institut Pasteur du Laos, Vientiane, Laos. 20 Current address: Symbiosis Technologies for Insect Control (SymbioTIC). Plateforme de 21 Recherche Cyroi, 2 rue Maxime Rivière, 97490 Ste Clotilde, France. 22 23 *Correspondence: 24 Julien Cattel, 25 Symbiosis Technologies for Insect Control (SymbioTIC) 26 Plateforme de recherche Cyroi, 2 rue Maxime Rivière, 97490 Ste Clotilde, France 27 E-mail: juliencattel@gmail.com 28 29 Email of all authors: 30 JC: juliencattel@gmail.com 31 CH: chloehbk@gmail.com 32 FL: frederic.laporte@univ-grenoble-alpes.fr 33 TG: thierry.gaude@univ-grenoble-alpes.fr 34 TC: t.cumer.sci@gmail.com 35 JR: julien.renaud@univ-grenoble-alpes.fr 36 IWS: ian.w.sutherland.mil@mail.mil 37 JCH: jeffrey.c.hertz.mil@mail.mil 38 JMB: jean-marc.bonneville@univ-grenoble-alpes.fr 39 VA: victor.arnaud19@gmail.com 40 CN: camille.nous@cogitamus.fr 41 BF: b.fustec@gmail.com 42 SB: sboyer@pasteur-kh.org 43 SM: s.marcombe@pasteur.la 44 JPD: jean-philippe.david@univ-grenoble-alpes.fr 45

mutation because of strong genetic constraints (Weill et al., 2004) suggest that CCE 97 amplifications play a central role in organophosphate resistance and are thus of high interest for 98 resistance monitoring. 99 In the tiger mosquito Aedes albopictus, the over-expression of two CCE genes (CCEae3A and 100 CCEae6A) through gene amplification was associated with resistance to the organophosphate 101 insecticide temephos (Grigoraki et al., 2017). In the yellow fever mosquito Aedes aegypti,  Further functional studies confirmed that CCEAE3A is able to sequester and metabolize the 106 active form of temephos in both Ae. aegypti and Ae. albopictus (Grigoraki et al., 2016). 107 Although the genomic structure and polymorphism of this CCE amplification was studied in 108 Ae. albopictus (Grigoraki et al., 2017) such work has not been conducted in Ae. aegypti. In 109 addition, the role of this CCE amplification in resistance to other insecticides remains unclear. 110 Finally, no high-throughput assay has yet been developed to track this resistance mechanism in 111 natural populations although such tool would significantly ease resistance monitoring and 112 management. 113 In this context, we combined an experimental evolution experiment with RNA-seq and 114 whole genome sequencing data to confirm the association between this genomic amplification, 115 the overexpression of CCE genes and resistance to organophosphate insecticides in Ae. aegypti. 116 We also showed that this CCE amplification confers resistance to multiple organophosphates  Table S1) and stored individually 140 at -20°C in silica gel until molecular analyses.  (Table S1). This population was then maintained for 2 145 generations without insecticide selection to allow genetic mixing before initiating insecticide was artificially selected with malathion at the adult stage for 4 consecutive generations (from 149 G1 to G5). For this, batches of thirty-three-days old non-blood fed adult mosquitoes (~1000 150 individuals of mixed sex) were exposed at each generation to filters papers impregnated with 151 malathion using WHO test tubes. A constant dose of 5% malathion coupled with an exposure 152 time of 10 min (leading to ~90% mortality at G1) were used through the whole selection 153 process. Surviving females were collected 48h after insecticide exposure, blood fed on mice 154 and allowed to lay eggs to generate the next generation.

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Three days-old non-blood fed adult females (not exposed to insecticide) were sampled after  Sampled mosquitoes were stored at -20°C until molecular analyses.

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Bioassays were used to monitor the dynamics of malathion resistance during the selection 163 process. Four replicates of 20 calibrated three days-old non-blood fed females not previously 164 exposed to insecticide were sampled at each generation and exposed to the insecticide as 165 described above using the same dose and exposure time as for artificial selection. Mortality was 166 recorded 48h after exposure.

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Cross resistance to other insecticide was investigated in G5 individuals (G5-Mala and G5-NS) 168 not previously exposed to insecticide. Calibrated individuals were exposed to three distinct 169 insecticides: the organophosphates fenitrothion and temephos, and the pyrethroid deltamethrin.

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For the adulticides fenitrothion and deltamethrin, bioassays were performed on eight replicates 171 7 of fifteen three days old non-blood-fed females with the following doses and exposure times: 172 fenithrotion 1% for 30 min, deltamethrin 0.05% for 20 min. Mortality rates were recorded 48h 173 after exposure. For the larvicide temephos, bioassays were performed on eight replicates of 174 twenty calibrated third instar larvae exposed to 0.08 mg/μL temephos for 24h in 200 ml tap 175 water and mortality was recorded at the end of exposure. then filtered based on their quality and alignment score as follows: mean read quality > 25, max 198 N allowed per read = 5, mapping quality ≥120, no multiple match allowed, read length ≥ 35.        (Table S3). Among them, 24 genes encoded proteins potentially 338 associated with known resistance mechanisms including cuticle alteration (14 genes) and 339 detoxification (10 genes). Only seven candidate genes were over-transcribed in the G5-Mala 340 line, all being associated with detoxification ( Figure 3A). This included a microsomal        Table S1.

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Cross comparing CNV data obtained for the CCE gene AAEL023844 between standard qPCR 474 and digital droplet PCR (ddPCR) indicated a good correlation between the two techniques 475 (r=0.85, P<0.001, Figure S1) suggesting that despite the technical variations inherent to qPCR 476 on single mosquitoes this approach provides a relatively good estimation of gene copy numbers.      Comparing gene copy number estimated from standard qPCR and TaqMan assays revealed a 537 good correlation between the two techniques (r=0.84, P<0.001) ( Figure 7B) although CNV 538 levels obtained with the TaqMan assay were lower as compared to those obtained with qPCR 539 and dd qPCR using primers targeting a different fragment.  Ae. albopictus (Grigoraki et al., 2017;Guillemaud et al., 1999;Qiao & Raymond, 1995).